Program Director/Principal Investigator (Last, First, Middle): Pielak, Gary J. Project Summary. Tardigrades are microscopic animals that survive extreme stresses, including desiccation, freezing, boiling, large doses of ionizing radiation and even the vacuum of outer space. We have identified the family of proteins that allows tardigrades to survive desiccation. These Cytosolic Abundant Heat Soluble (CAHS) proteins are unique to tardigrades. The goal of the project is to define the protective mechanism used by CAHS proteins to shield client proteins from desiccation-induced damage. This knowledge will facilitate the development of new molecules to increase the stability and shelf life of protein-based drugs and industrial enzymes. We hypothesize that CAHS proteins protect client proteins via encapsulation in a reversible matrix. We propose a model where fully hydrated CAHS proteins possess partially-collapsed termini separated by a flexible linker. Upon water removal, the termini interact intermolecularly, forming a protective gel matrix around the client proteins. The matrix persists and maintains client protein protection even upon complete desiccation. The model is supported by preliminary published and new data from studies of enzyme activity, NMR-detected amide proton exchange, 19F NMR, circular dichroism spectropolarimetry, Fourier-transform infrared spectroscopy and rheometry. The model will be further tested in three independent and complementary aims. Aim 1: Determine sequence/function relationships in the solid state. We predict that protection is modular; linkers can be swapped, and ends can be swapped as long as each molecule possesses two ends separated by a linker. We will test this idea using fragments comprising the modules alone and combinations of modules. We will determine the degree to which CAHS proteins and their fragments protect enzymes against desiccation- induced inactivation and assess their ability to preserve the structure of a model protein in the solid state by using NMR-detected amide proton/deuterium exchange. Aim 2: Determine sequence/function relationships in gels and solution. We will quantify the effects of CAHS proteins and the fragments on their modified standard-state free energy of unfolding of a client protein. Aim 3: Define sequence/structure relationships in the solid state, gels and solution. We will quantify the ability of CAHS proteins and their fragments to form gels using rheometry, a method to quantify gel strength. The secondary structure of intact CAHS proteins and their fragments will be quantified using synchrotron-radiation circular-dichroism spectropolarimetry and Fourier-transform infrared spectroscopy. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page